Carmen María Salvador Ferrer

Validation and uncertainty study of a comprehensive list of 160 pesticide residues in multi-class vegetables by liquid chromatography–tandem mass spectrometry

A rapid and sensitive liquid chromatography-tandem mass spectrometry method, in electrospray ionization positive mode, has been developed for the determination of 160 selected multi-class pesticides over a 33-min run time. Extracts were obtained using the acetonitrile-based QuEChERS (quick, easy, cheap, effective, rugged and safe) sample preparation technique. The validation study was carried out on tomato, pear and orange matrices following DG SANCO/2007/3131 of the European Quality Control Guidelines. These matrices represent high water, high sugar and high acidic content commodities, respectively. Matrix influence on recoveries and its effects on ionization were evaluated for the three matrices. Ten out of the 160 pesticides showed very low intensity, linearity and/or sensitivity problems. Linearity was studied in the 5-500 microg kg(-1) concentration range. Soft (<20%), medium (20-50%), and strong (>50%) matrix effects were obtained for 69%, 20%, and 11% of the studied compounds, respectively. Recoveries were investigated at the 10 and 100 microg kg(-1) levels, and depending on the commodity, 97%, 98% and 97% of the compounds in tomato, pear and orange, respectively, were in the 70-120% range. More than 90% of the investigated compounds had less or equal to a 5 microg kg(-1) limit of detection in the studied matrices. The relative standard deviations obtained exceeded 20% in only very few cases. The overall standard deviation obtained in the survey study (0.1551) was used for the methods uncertainty estimation. The expanded uncertainty resulted as being 0.3002 (coverage factor K=2, confidence level 95%). The method was applied on 59 real samples from 14 different kinds of fruits and vegetables. Thirty-three compounds were detected in 50 positive samples.

Overcoming matrix effects using the dilution approach in multiresidue methods for fruits and vegetables.

During recent years matrix effects in liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) have quickly become a major concern in food analysis. The phenomenon of ion suppression can lead to errors in the quantification of the analytes of interest, as well as can affect detection capability, precision, and accuracy of the method. Sample dilution is an easy and effective method to reduce interfering compounds, and so, to diminish matrix effects. In this work, matrix effects of 53 pesticides in three different matrices (orange, tomato and leek) were evaluated. Several dilutions of the matrix were tested in order to study the evolution of signal suppression. Dilution of the extracts led to a reduction of the signal suppression in most of the cases. A dilution factor of 15 demonstrated to be enough to eliminate most of the matrix effects, opening the possibility to perform quantification with solvent based standards in the majority of the cases. In those cases where signal suppression could not be reduced, a possible solution would be to use stable isotope-labelled internal standards for quantification of the problematic pesticides.

Liquid chromatography-high-resolution mass spectrometry for pesticide residue analysis in fruit and vegetables: Screening and quantitative studies

This work reviews the current state-of-the-art of liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) techniques applied to the analysis of pesticides in fruit-based and vegetable-based matrices. Nowadays, simultaneous trace analysis of hundreds of pesticides from different classes is required, preferably in just one run. The most commonly used QqQ-MS technology presents certain limitations in its application in a cost and effective way when analyzing a large number of pesticides. Thus, this review includes HRMS technology as a reliable complementary alternative allowing the analysis of a wide range of pesticides in food. Its capabilities and limitations in identifying, confirming and quantifying pesticides are discussed. HRMS instruments can adequately address such issues; however, the main drawbacks are as a result of insufficient prior optimization of the operational parameters during non-target analysis in full-scan mode and due to software shortcomings.

Determination of pesticide residues in high oil vegetal commodities by using various multi-residue methods and clean-ups followed by liquid chromatography tandem mass spectrometry.

Several extraction methods were evaluated in terms of recoveries and extraction precision for 113 pesticides in avocado: QuEChERS with various d-SPE clean-ups (Z-Sep, Z-Sep+, PSA+C18 and silica), miniLuke and ethyl acetate. Extracts were analysed using liquid chromatography coupled with triple quadrupole mass spectrometer working in multi-reaction monitoring mode. Z-Sep and Z-Sep+ are new types of material for high lipid matrices - these two sorbents contain ZrO2, which improves fat removal from the extracts. The QuEChERS protocol with Z-Sep provided the highest number of pesticides with recoveries in the 70-120% range along with the lowest amount of coextracted matrix compounds. Subsequently, this method was validated in two matrices - avocado and almonds. In the validation recoveries at two levels - 10 and 50μg/kg - limit of quantitation, linearity, matrix effects, as well as the inter- and intraday precision were studied. In the avocado samples, 107 analytes had LOQs equal to 10μg/kg (signal to noise of quantitative transition was equal 20 or more). In the almond samples, 92 pesticides had LOQs equal to 10μg/kg (S/N≥20) and 2 pesticides at 50μg/kg. The validated method was employed in the analysis of real avocado and almond samples.

Pesticide residue analysis of fruit juices by LC-MS/MS direct injection. One year pilot survey.

For this work, thirteen types of fruit juices (orange, pineapple, peach, apple, multifruit, mango, strawberry, tomato, pear, mandarin, grape, banana and grapefruit) were selected to develop an analytical method for the analysis of 53 pesticides by direct injection in LC-MS/MS. The preparation of the samples was very simple: an aliquot of the juice was centrifuged and it was ten-times diluted prior to analysis, which allowed reducing considerably the time and cost of the analyses. Besides, dilution of the samples permits reducing the amount of matrix going into the system, and thus, decreasing the matrix effects, so common in this type of commodities, opening the possibility to perform quantification with solvent based standards. Validation of the method was carried out in accordance with EU guidelines. Calibration curves covering three orders of magnitude were performed, and they were linear over the concentration range studied for all the matrices (from 0.1 to 100 μg L(-1)). Practical limits of quantification were in the low μg L(-1) range, far below the maximum residue levels (MRLs) of the EU regulations, which do not set specific MRLs for juices, and in this cases of processed food, MRLs of the raw product are applied. Repeatability of the instrumental method was studied in all matrices, obtaining good intra- and inter-day relative standard deviations (RSDs). The proposed method was applied to 106 real fruit juice samples purchased in different local markets during a one-year survey in order to validate the suitability for routine analysis. 43% of the analysed samples gave positive results (higher than the practical limits of quantification).

Study of the effects of operational parameters on multiresidue pesticide analysis by LC-MS/MS

In this paper, the influence of several operational parameters on a well established multiresidue LC-MS/MS method has been studied in relation to the analysis of 150 pesticides commonly present in vegetable samples. The operational parameters investigated are: (i) the influence of different modifiers (0.1% formic acid; 5mM ammonium formiate; 5mM ammonium acetate in aqueous phase) - both on the retention time and on the analytical response of the studied compounds; (ii) the effect of the analytical columns temperature on the retention time and on the analytical response of the pesticides investigated; (iii) the effects of co-elution in mixture containing 150 pesticides and, additionally, (iv) the carrying out of a study about the common transitions obtained by LC-MS/MS. Various common transitions were found among the 150 pesticides, but there were only two problematic cases, the pairs diuron-fluometuron and prometryn-terbutryn, which have common scanned transitions and have very close retention times. The use of ammonium salts as modifier instead of formic acid reports enhancement or suppression of the response depending on the pesticides. No great influence on the retention time or on the response of the pesticides and commodities studied was observed with relation to the column temperature. Two different columns: an HPLC (5 μm particle size) and an UHPLC analytical column (1.8 μm particle size) have been used. As was expected, shorter run times and lower peak width was achieved with the UHPLC column. In this paper, the effect of the compounds on each other in the MS analysis when the number of co-eluting compounds is quite high is also described. Mainly small suppression or enhancement co-elution effect was observed, but some particular pesticides presented high sensitivity (> ± 60% effect) when they elute together with others. This is an important factor and it has to be taken into account when performing multiresidue pesticide analysis.

Determination of selected non‐authorized insecticides in peppers by liquid chromatography time‐of‐flight mass spectrometry and tandem mass spectrometry

In this work, two analytical methods based on liquid chromatography coupled to electrospray time-of-flight mass spectrometry (LC/ESI-TOFMS) and tandem mass spectrometry (LC/ESI-MS/MS) are described for the identification, confirmation and quantitation of three insecticides non-authorized in the European Union (nitenpyram, isocarbophos and isofenphos-methyl) but detected in recent monitoring programmes in pepper samples. The proposed methodologies involved a sample extraction procedure using liquid-liquid partition with acetonitrile followed by a cleanup step based on dispersive solid-phase extraction. Recovery studies performed on peppers spiked at different fortification levels (10 and 50 microg kg(-1)) yielded average recoveries in the range 76-100% with relative standard deviation (RSD) (%) values below 10%. Identification, confirmation and quantitation were carried out by LC/TOFMS and LC/MS/MS using a hybrid triple quadrupole linear ion trap (QqLIT) instrument in multiple-reaction monitoring (MRM) mode. The obtained limits of quantitation (LOQs) were in the range 0.1-5 microg kg(-1), depending on each individual technique. Finally, the proposed methods were successfully applied to the analysis of suspected pepper samples.

Analysis of forchlorfenuron in vegetables by LC/TOF-MS after extraction with the buffered QuEChERS method.

This paper describes the application of liquid chromatography time-of-flight mass spectrometry (LC/TOF-MS), with electrospray ionization, for residue analysis of forchlorfenuron in tomato, zucchini and watermelon. The assessed method includes a sample preparation step based on the buffered QuEChERS approach. The TOF-MS fragmentation pattern of forchlorfenuron was studied at different fragmention voltages in the range of 120-270 V. Analyses were carried out under full-scan conditions by using the extracted ion chromatogram (XIC) of the m/z 248 ion with a 0.2 Da window. The linearity of the analytical response across the studied range of concentrations (10-500 microg/kg) was excellent, obtaining correlation coefficients higher than 0.999, and relative standard deviations of the response factors lower than 14%, for the 15 linear calibration curves of forchlorfenuron evaluated along the complete validation study. No significant matrix effects were observed. The signal-to-noise ratios obtained for the 10 microg/kg forchlorfenuron in matrix matched standards were >70 for all three matrices. Recovery studies were carried out on spiked tomato, zucchini and watermelon blank samples, at three concentration levels (10, 50, and 200 microg/kg) performing five replicates at each level. Forchlorfenuron mean recoveries ranged between 80% and 87% in watermelon and zucchini, and between 65% and 71% in tomato, obtaining in all cases relative standard deviation values lower than 10%. The method readily achieved a lowest validated level of 10 microg/kg, which was fit-for-purpose in residue monitoring applications.

Processing factor for a selected group of pesticides in a wine-making process: distribution of pesticides during grape processing

The processing factors (the pesticide concentration found in the wine/pesticide concentration found in grapes) of acetamiprid, azoxistrobin, carbaril, carbendazime, cyprodinil, dimethoate, dimethormorf, imazalil, imidacloprid, kresoxim methyl, penconazole, procymidone and thiabendazole were determined in a wine-making process. Pesticide analysis was performed using a multi-residue method for the determination of different pesticides both in wine and in grapes by extraction with acetonitrile followed by LC/MS. The pesticide distribution was studied for each step of the process, and the pesticide processing factors were calculated and found to vary among the different pesticides studied. pKow was found to affect a pesticide’s processing factor; a linear correlation was obtained for all pesticide processing factors, except for dimethoate, which was the most water soluble. However, no correlation was found between the processing factor and the water solubility of pesticides.

Analysis of illegal dyes in food by LC/TOF-MS

A sensitive and accurate methodology was developed for the analysis of seven illegal dyes (Sudan I, Sudan II, Sudan III, Sudan IV, Sudan Orange G, Sudan R and Para Red,) used as additives in food products, such as chilli powder and steak sauces. The analytical methodology consisted of solvent extraction with acetonitrile followed by liquid chromatography time-of-flight mass spectrometry detection. Accurate mass measurements were crucial in order to achieve a high degree of specificity for the target analytes in such complex samples. The dyes were effectively extracted from spice and sauce matrices achieving recoveries higher than 75%. Because of the excellent mass accuracy obtained for the target analytes (better than 2 ppm), no cleanup of the samples was required using this methodology, thus leading to a better precision and reproducibility of the results from the quantitative point of view. Calibration curves were linear and covered two orders of magnitude (from 0.01 to 1 mg L−1) for all the compounds studied with the exception of Para Red. A detailed study of matrix effects is also included in this work, showing a clear improvement when dilution of the extracts was carried out. Method detection limits were in the low mg kg−1 range, and the precision, calculated as the relative standard deviation, ranged from 5 to 15%. The methodology was successfully applied to market samples in a survey performed as part of a regional research programme organized by the Andalusian Health Service in Spain, and a positive confirmation for Sudan I was obtained in a chilli powder sample.